Furthermore, compounds B4 and D4 exhibited lower hepatotoxicity than tacrine in cell viability, apoptosis, and intracellular ROS production for HepG2 cells. These properties of compounds B4 and D4 suggest that they deserve further investigation as promising agents for the prospective treatment of AD.A series of OA-tacrine hybrids with the alkylamine linker was designed, synthesized, and evaluated as effective cholinesterase inhibitors for the treatment of Alzheimer’s disease (AD). Biological activity results demonstrated that some hybrids possessed significant inhibitory activities against acetylcholinesterase (AChE). Among them, compounds B4 (  Acetylcholinesterase inhibitors (AChEIs), the first drugs used in the treatment of AD, can enhance the concentration of ACh in the synaptic cleft and improve behavioral disorders in AD patients. Five AChEIs  have bee,,,The research of traditional Chinese medicine (TCM) in China has a long history and resource advantage. TCM treatment is characterized by integrity and multi-targets. Therefore, starting from natural active ingredients, is very beneficial to the treatment of AD with complex and multiple etiologies. Many natural products have significant therapeutic effects on AD, such as flavonoids, coumarins, alkaloids, phenylpropanoids, triterpenoid saponins, etc. Oleanolic acid OA, , a bioacIt was demonstrated by X-ray crystallography that AChE contains two binding sites: the catalytic active site (CAS) and the peripheral anionic site (PAS) connected by a 20 Å deep hydrophobic gorge.3a–g were obtained from commercial methyl anthranilate by hydrolysis, condensation, and chlorination reactions. And then, target compounds A1–G5 were synthesized via the reaction of diamine intermediates 4a1-4g5 and the commercial OA. The structures of all newly synthesized compounds were confirmed by various methods of spectroscopic analysis, such as 1H NMR, 13C NMR, and ESI-MS.As shown in hAChE and hBuChE inhibitory activities of OA-tacrine hybrids were measured by Ellman’s method using tacrine as reference compound.50 values and selectivity index (SI) values are shown in 50 values of B1-4, D1-5, and F1-5, compounds with Cl or Br substitution from the benzene ring of tacrine performed much better activities than that with F substitution in hAChE inhibition. Among them, compound D4  with Br substitution showed the most potent inhibitory activity and selectivity for hAChE, which was stronger than the reference compound tacrine . Compounds B4  and D4 with R1-position substitution had more potent hAChE inhibitory activities than compounds C5  and E4  with R2-position substitution. Compounds G1-5 with R2-position substitution had more potent hBuChE inhibitory activities. Among them, G1  with F substitution showed the most potent inhibitory activity and selectivity for hBuChE.The To gain insights into the binding patterns of these compounds with the AChE enzymes, the molecular modeling study based on AChE (PDB code: 2CKM) was performed using the docking program, AutoDock 4.2 package with Discovery Studio 2.0. As shown in hAChE inhibitory activities, were selected as representative compounds to be evaluated for potential cytotoxic effects at the concentrations of 25, 50, 100 μM. OA and tacrine were used as the reference. As shown in The safety is extraordinarily important for the CNS drugs, so the potential toxicity effect of OA-tacrine hybrids was investigated on SH-SY5Y cells and HepG2 cells by Cell Counting Kit-8 (CCK-8) assay. Compounds B1, B4, and D4, which had more potent As mentioned above, hepatotoxicity is a major side effect for tacrine. Considering hepatoprotective efficacy of OA, the strategy adopted for the design of OA-tacrine hybrids in this work. To further investigate the hepatotoxicity of hybrids, AnnexinV/PI double staining method was used to evaluate the effects of compounds B4 and D4 on cell apoptosis at the concentration of 50 μM. As shown in ,.Studies have shown that the hepatotoxicity induced by tacrine mainly resulted in elevating the levels liver transaminase, decreasing albumin concentration and inducing ROS in hepatocyte hAChE, IC50 = 14.37 ± 1.89 nM; SI > 695.89) and D4  showed more potent inhibitory activities and selectivities for hAChE, which were stronger than the reference compound tacrine . The molecular modeling study has been done to gain insights into the binding patterns of these compounds with the AChE enzymes. More importantly, the cytotoxicity tests showed that compounds B4 and D4 had low cytotoxic activity toward SH-SY5Y cells and HepG2 cells. Besides, these compounds showed lower hepatotoxicity in HepG2 cells by inducing less cell apoptosis and intracellular ROS than tacrine. These properties highlighted that compounds B4 and D4 with high AChE inhibitory activities, low neurotoxicity and hepatotoxicity could be considered as potential agents for the development of anti-AD drugs.In summary, a series of OA-tacrine hybrids with the alkylamine linker was designed and synthesized as effective ChEs inhibitors against AD. Among the synthesized compounds, compounds B4 . 1H NMR and 13C NMR spectra were measured on a Bruker ACF-500 spectrometer at 25 °C and referenced to TMS. Chemical shifts are reported in ppm (δ) using the residual solvent line as internal standard. Splitting patterns are designed as s, singlet; d, doublet; t, triplet; m, multiplet. Mass spectra were obtained on a MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI-MS).All chemicals (reagent grade) used were purchased from Sino pharm Chemical Reagent Co., Ltd. (China). Reaction progress was monitored using analytical thin layer chromatography (TLC) on precoated silica gel GF254  plates and the spots were detected under UV light (254 nm). Column chromatography was performed on silica gel (90–150 1a–g (5.0 mmol) were dissolved in 20% NaOH aqueous solution stirred for 5 h, adjusted the pH to 2, filtered and dried to obtain anthranilic acid 2a–g. Then under ice bath (0 °C), slowly add compound 2a–g (5.0 mmol) and cyclohexanone (6.0 mmol) to POCl3 (15 ml). 5 min later, removed the ice bath and stirred at 90 °C with refluxing for 5 h, adjusted the pH to 10, extracted with dichloromethane and subjected to column chromatography (petroleum ether: ethyl acetate = 10: 1).Commercial methyl anthranilate 1H NMR  δ 8.12 , 7.94 , 7.75 , 7.64 , 3.04 , 2.96 , 1.88 ; ESI-MS m/z 218.1[M + H]+.White solid , yield 82.00%. 1H NMR  δ 8.08 , 7.76 , 7.42 , 3.20 , 3.00 , 1.95 ; ESI-MS m/z 252.1[M + H]+.Yellow solid , yield 78.03%. 1H NMR  δ 7.29 − 7.23 , 7.06 , 6.93 , 2.78 , 2.15 − 2.06 , 1.95 − 1.88 , 1.41 ; ESI-MS m/z 252.1[M + H]+.Yellow solid , yield 74.10%. 1H NMR  δ 8.18 , 8.01 , 7.60 , 3.12 , 2.98 , 1.95 ; ESI-MS m/z 295.9[M + H]+.Yellow solid , yield 86.03%. 1H NMR  δ 8.15 , 7.99 , 7.36 , 3.24 − 3.17 , 3.07 − 2.99 , 1.96 ; ESI-MS m/z 295.9[M + H]+.Yellow solid , yield 83.97%. 1H NMR  δ 8.15 , 7.60 , 7.31 , 3.10 , 2.99 , 1.94 ; ESI-MS m/z 236.1[M + H]+.Brown solid , yield 70.02%. 1H NMR  δ 7.93 , 7.45 , 7.35 , 3.18 , 3.02 , 1.95 ; ESI-MS m/z 236.1[M + H]+.Brown solid , yield 64.03%. 3a–g (1.0 mmol), diaminoalkanes (6.0 mmol) of various lengths  and KI (0.1 mmol) were mixed and dissolved in ethylene glycol (10 ml). Heat up to 160 °C and stirred for 8h, then extracted with dichloromethane and concentrated to obtain brown oil diamine intermediates 4a1–4g5. Meanwhile, oleanolic acid (1.1 mmol) was dissolved in THF with HATU (1.1 mmol) and DIPEA (2.2 mmol) at room temperature and stirred for 0.5h. Then added brown oil diamine intermediates 4a1–4g5 to them. About 3-5h later, concentrated to column chromatography (dichloromethane: Methanol = 20: 1).Tacrine derivatives 1H NMR  δ 12.18 , 8.23 , 7.87 , 7.83 , 7.69 , 7.51 − 7.46 , 5. 46 , 3.97 , 3.64 , 3.44 , 3.26 − 3.18 , 3.12 , 3.02 , 2.63 , 2.59 , 2.08 − 1.98 , 1.95 − 1.90 , 1.86 , 1.76 , 1.73 − 1.66 , 1.58 − 1.52 , 1.44 , 1.25 , 1.14 , 0.97 , 0.90 , 0.81 , 0.75 , 0.70 ; 13C NMR  δ 156.22, 150.44, 143.38, 138.33, 132.82 (C6-Tacrine), 129.84 (C5-Tacrine), 129.80 (C7-Tacrine), 125.35 (C8-Tacrine), 124.72, 123.72, 119.79, 115.50, 111.43, 54.94, 54.84, 47.77, 47.32, 46.36, 46.33, 43.00, 41.80, 41.51, 39.28, 38.64, 38.31, 36.80, 33.90, 32.84, 32.21, 30.52, 29.63, 28.19, 27.98, 27.60, 27.13, 25.72, 23.39, 23.36, 21.74, 20.59, 18.40, 18.15, 16.97, 16.85, 15.50, 15.14, 12.47; ESI-MS m/z 680.5 [M + H]+.White solid , yield 32.33%. 1H NMR  δ 10.74 , 8.28 , 8.19 , 7.82 , 7.69 , 7.46 , 5.44 , 4.16 − 4.07 , 3.85 − 3.59 , 3.19 , 3.03 , 2.74 − 2.66 , 2.66 − 2.60 , 2.08 − 2.00 , 1.95 − 1.91 , 1.88 , 1.74 , 1.59 − 1.56 , 1.54 − 1.51 , 1.23 − 1.18 , 1.14 , 0.96 , 0.90 , 0.78 , 0.74 , 0.55 ; 13C NMR  δ 182.29, 157.03, 143.70(C6-Tacrine), 132.95(C5-Tacrine), 125.20(C7-Tacrine), 123.50(C8-Tacrine), 119.49, 115.56, 111.43, 55.72, 47.31, 46.44, 46.28, 43.67, 41.81, 41.61, 39.67, 39.20, 38.63, 38.30, 36.80, 33.93, 32.87, 32.61, 32.13, 30.60, 29.62, 29.58, 29.24, 28.22, 27.94, 27.16, 27.13, 26.98, 23.49, 23.41, 23.33, 21.89, 20.69, 18.48, 18.06, 17.02, 16.54, 15.41, 15.11, 14.05, 12.59; ESI-MS m/z 708.5 [M + H]+.White solid , yield 35.29%. 1H NMR  δ 11.30 , 8.25 , 7.82 , 7.72 , 7.49 , 6.15 , 5.38 , 3.92 , 3.71 , 3.35 , 3.24 − 3.17 , 3.06 − 3.02 , 3.00 , 2.63 , 2.55 , 1.93 , 1.91 − 1.87 , 1.75 , 1.68 − 1.58 , 1.57 − 1.51 , 1.43 , 1.43 − 1.38 , 1.36 , 1.27 − 1.22 , 1.20 − 1.18 , 1.15 , 0.98 , 0.90 , 0.86 , 0.75 ; 13C NMR  δ 156.29, 150.06, 144.45, 138.19, 132.99, 125.31(C6-Tacrine), 124.95(C5-Tacrine), 122.87(C7-Tacrine), 119.57(C8-Tacrine), 115.37, 111.32, 55.62, 54.93, 48.03, 47.35, 46.54, 46.15, 43.58, 41.98, 41.91, 39.22, 38.98, 38.62, 38.30, 36.81, 33.99, 32.85, 32.63, 32.27, 30.58, 30.20, 29.01, 28.21, 27.98, 27.12, 26.96, 25.95, 25.63, 25.58, 23.68, 23.44, 23.37, 22.88, 21.61, 20.52, 18.40, 18.14, 16.88, 15.49, 15.20, 12.68; ESI-MS m/z 722.5 [M + H]+.White solid , yield 51.21%. 1H NMR  δ 11.47 , 8.24 , 7.90 , 7.73 − 7.68 , 7.51 − 7.44 , 6.07 , 5.38 , 3.93 , 3.40 , 3.21 , 3.04 , 2.61 , 2.56 − 2.50 , 2.00 , 1.92 , 1.90 − 1.86 , 1.75 , 1.70 − 1.64 , 1.60 , 1.59 − 1.57 , 1.55 , 1.54 − 1.50 , 1.47 , 1.45 , 1.36 , 1.33 , 1.27 − 1.24 , 1.23 , 1.19 , 1.15 , 1.08 − 1.00 , 0.98 , 0.95 , 0.89 , 0.86 , 0.75 ; 13C NMR  δ 178.95, 156.39, 150.62, 144.68, 138.30, 133.14, 125.45(C6-Tacrine), 124.81(C5-Tacrine), 122.95(C7-Tacrine), 120.00(C8-Tacrine), 115.53, 111.28, 55.03, 53.43, 50.87, 48.73, 47.46, 46.71, 46.33, 42.13, 42.04, 39.35, 38.87, 38.73, 38.40, 36.92, 34.11, 32.95, 32.73, 32.33, 31.92, 31.90, 30.70, 30.01, 29.77, 29.69, 29.65, 29.60, 29.55, 29.51, 29.48, 29.36, 29.34, 29.30, 29.23, 28.31, 28.08, 27.26, 27.20, 27.09, 25.74, 24.03, 23.83, 23.56, 23.48, 23.21, 22.68, 21.73, 20.59, 18.25, 16.99, 15.57, 15.32, 14.12; ESI-MS m/z 736.5 [M + H]+.White solid , yield 27.51%. 1H NMR  δ 7.99 − 7.95 , 7.92 , 7.28 , 6.00 , 5.30 , 3.57 , 3.43 − 3.36 , 3.20 , 3.07 , 3.04 , 2.68 , 2.47 , 1.96 , 1.91 , 1.84 , 1.75 , 1.68 , 1.63 − 1.60 , 1.61 − 1.58 , 1.57 , 1.55 , 1.53 − 1.50 , 1.44 , 1.25 , 1.15 , 1.03 , 0.98 , 0.89 , 0.84 , 0.77 , 0.70 ; 13C NMR  δ 178.61, 145.02(C6-Tacrine), 124.67(C5-Tacrine), 124.59(C7-Tacrine), 122.79(C8-Tacrine), 55.06, 48.76, 47.47, 46.77, 46.32, 42.30, 42.10, 39.35, 38.93, 38.76, 38.45, 36.93, 34.13, 32.95, 32.61, 32.31, 30.72, 29.71, 29.33, 28.95, 28.09, 27.27, 27.22, 27.14, 27.05, 25.71, 24.66, 23.87, 23.56, 23.50, 22.80, 22.34, 18.27, 16.97, 15.57, 15.33, 14.13; ESI-MS m/z 714.5 [M + H]+.White solid , yield 30.79%. 1H NMR  δ 12.48 , 8.16 , 7.92 , 7.32 , 6.31 , 5.41 , 3.96 , 3.42 , 3.21 , 3.15 , 3.09 , 2.63 , 2.58 , 2.01 , 1.92 − 1.88 , 1.86 , 1.75 , 1.72 − 1.64 , 1.64 − 1.54 , 1.53 − 1.49 , 1.25 , 1.22 − 1.18 , 1.16 , 0.98 , 0.90 , 0.87 , 0.76 ; 13C NMR  δ 179.34, 156.02, 151.21, 144.41(C6-Tacrine), 139.02, 138.83, 126.62(C5-Tacrine), 126.02(C7-Tacrine), 123.14(C8-Tacrine), 118.65, 113.89, 111.73, 55.08, 47.92, 47.51, 46.67, 46.32, 42.02, 41.96, 39.39, 38.76, 38.69, 38.44, 36.96, 34.15, 32.98, 32.84, 32.39, 30.71, 29.71, 29.35, 29.33, 28.43, 28.10, 27.54, 27.30, 27.22, 27.14, 26.84, 25.78, 23.81, 23.56, 23.52, 23.49, 21.74, 20.54, 18.28, 17.04, 15.59, 15.33, 14.13; ESI-MS m/z 742.5 [M + H]+.White solid , yield 25.59%. 1H NMR  δ 10.13 , 8.30 , 7.98 , 7.38 , 6.86 , 5.37 − 5.33 , 4.11 − 4.00 , 3.69 − 3.66 , 3.64 − 3.61 , 3.16 , 3.12 , 3.06 , 2.85 , 2.62 , 1.97 , 1.87 , 1.77 , 1.65 , 1.60 − 1.51 , 1.49 , 1.06 , 0.88 , 0.83 , 0.67 , 0.39 ; 13C NMR  δ 182.22, 165.55, 157.70, 143.32(C6-Tacrine), 136.61(C5-Tacrine), 125.69(C7-Tacrine), 123.47(C8-Tacrine), 112.34, 78.71, 77.37, 77.16, 76.95, 55.60, 55.49, 55.36, 54.98, 52.15, 47.34, 46.37, 46.20, 43.44, 41.71, 41.34, 41.20, 39.62, 39.20, 38.67, 38.63, 38.63, 38.59, 38.56, 38.34, 37.95, 36.82, 33.96, 32.95, 32.77, 32.25, 31.83, 30.60, 29.62, 29.24, 28.74, 28.02, 27.21, 27.01, 25.82, 25.70, 23.45, 23.30, 23.26, 21.71, 20.68, 18.40, 18.27, 18.15, 17.00, 16.89, 16.76, 16.52, 15.60, 15.54, 15.12, 12.71; ESI-MS m/z 756.5 [M + H]+.White solid , yield 35.69%. 1H NMR  δ 8.33 , 8.19 , 7.34 , 6.08 , 5.36 , 3.87 , 3.40 , 3.22 , 3.19 , 3.06 − 3.01 , 2.66 − 2.62 , 2.53 − 2.48 , 1.97 , 1.92 , 1.89 , 1.75 , 1.71 − 1.64 , 1.60 , 1.58 − 1.53 , 1.51 , 1.47 , 1.45 , 1.37 − 1.32 , 1.28 − 1.21 , 1.18 , 1.15 , 1.03 , 0.98 , 0.89 , 0.87 , 0.75 ; 13C NMR  δ 178.78, 155.25, 144.85(C6-Tacrine), 126.10(C5-Tacrine), 125.87(C7-Tacrine), 122.84(C8-Tacrine), 114.47, 111.65, 55.04, 50.84, 48.69, 47.47, 46.75, 46.32, 45.91, 42.18, 42.06, 39.35, 38.75, 38.43, 36.94, 34.12, 32.95, 32.70, 32.33, 30.71, 30.23, 29.69, 29.42, 28.08, 27.27, 27.13, 25.73, 24.08, 23.85, 23.75, 23.58, 23.50, 21.92, 20.72, 18.26, 16.98, 15.58, 15.35, 14.12, 8.65; ESI-MS m/z 770.5 [M + H]+; HRMS(ESI) m/z [M + H]+ calcd for C49H73N3ClO2+ 770.5391, found 770.5406.White solid , yield 35.04%. 1H NMR  δ 8.69 , 8.38 , 7.40 , 5.34 , 3.22 , 3.11 , 2.98 − 2.91 , 2.28 , 2.18 , 2.13 − 2.05 , 2.04 − 1.98 , 1.87 , 1.76 , 1.61 , 1.57 − 1.54 , 1.54 − 1.47 , 1.45 − 1.42 , 1.42 − 1.39 , 1.35 , 1.27 , 1.25 , 1.22 , 1.00 , 0.98 , 0.96 , 0.90 , 0.87 , 0.85 , 0.79 , 0.75 ; 13C NMR  δ 173.29, 151.48(C5-Tacrine), 142.07, 140.79, 134.95(C6-Tacrine), 129.17(C7-Tacrine), 123.66(C8-Tacrine), 120.56, 55.20, 47.56, 47.44, 45.54, 41.87, 41.59, 39.43, 38.70, 38.47, 36.95, 33.64, 32.88, 32.73, 32.11, 30.56, 29.62, 28.05, 28.03, 27.12, 25.66, 23.46, 23.40, 23.25, 18.29, 16.87, 15.51, 15.33; ESI-MS m/z 714.5 [M + H]+.Yellow solid , yield 46.18%. 1H NMR  δ 8.69 , 8.38 , 7.40 , 5.34 , 3.22 , 2.97 − 2.91 , 2.28 , 2.18 , 2.13 − 2.06 , 2.01 , 1.87 , 1.76 , 1.63 − 1.58 , 1.58 − 1.54 , 1.50 , 1.47 − 1.45 , 1.43 , 1.41 , 1.35 , 1.28 − 1.26 , 1.25 , 1.22 , 1.10 , 1.00 , 0.98 , 0.96 , 0.90 , 0.85 , 0.79 , 0.75 ; 13C NMR  δ 172.34, 150.54(C5-Tacrine), 141.13, 139.84, 134.01, 128.23(C6-Tacrine), 122.72(C7-Tacrine), 119.62(C8-Tacrine), 54.26, 46.62, 46.50, 44.60, 40.93, 40.65, 38.49, 37.76, 37.53, 37.13, 36.01, 32.70, 31.94, 31.79, 31.17, 30.22, 29.62, 28.68, 27.11, 27.09, 26.18, 24.72, 22.52, 22.46, 22.31, 17.35, 15.93, 14.57, 14.39; ESI-MS m/z 728.4 [M + H]+.Yellow solid , yield 23.34%. 1H NMR  δ 8.17 , 7.76 , 7.41 , 6.22 , 5.37 , 3.88 , 3.37 , 3.19 , 3.13 − 3.09 , 3.07 , 2.69 , 2.59 − 2.53 , 1.98 , 1.91 , 1.89 − 1.85 , 1.82 , 1.73 , 1.64 , 1.55 , 1.52 , 1.43 , 1.24 , 1.14 , 1.08 − 1.00 , 0.96 , 0.88 , 0.84 , 0.75 , 0.71 ; 13C NMR  δ 177.81, 154.97, 151.51, 143.46(C5-Tacrine), 135.24, 131.02(C6-Tacrine), 123.99(C7-Tacrine), 123.85, 123.09, 121.96(C8-Tacrine), 116.65, 112.64, 54.06, 52.43, 47.44, 46.49, 45.65, 45.24, 40.96, 40.90, 38.34, 37.75, 37.73, 37.42, 35.93, 33.15, 31.99, 31.76, 31.39, 29.69, 28.89, 28.68, 27.08, 26.71, 26.28, 26.12, 25.78, 24.75, 22.70, 22.57, 22.45, 20.84, 19.95, 17.26, 16.00, 14.58, 14.29; ESI-MS m/z 742.5 [M + H]+.Yellow solid , yield 33.67%. 1H NMR  δ 8.16 , 7.76 , 7.40 , 6.06 , 5.36 , 3.80 , 3.73 , 3.35 , 3.20 , 3.11 , 3.02 , 2.68 , 2.53 , 1.94 − 1.90 , 1.88 , 1.85 − 1.83 , 1.81 , 1.74 , 1.67 − 1.61 , 1.59 , 1.56 , 1.53 , 1.51 , 1.42 , 1.20 , 1.14 , 1.05 − 0.99 , 0.97 , 0.89 , 0.86 , 0.76 , 0.73 ; 13C NMR  δ 177.49, 154.42, 152.25, 143.72(C5-Tacrine), 136.11, 130.63, 124.88(C6-Tacrine), 123.62, 122.96(C7-Tacrine), 121.85(C8-Tacrine), 117.09, 112.95, 97.44, 66.96, 66.41, 54.06, 48.05, 46.50, 45.72, 45.25, 41.09, 41.02, 38.34, 38.01, 37.73, 37.43, 35.93, 33.15, 32.28, 31.98, 31.68, 31.37, 30.91, 29.70, 29.45, 29.18, 28.68, 28.64, 28.35, 28.17, 27.40, 27.08, 26.28, 26.13, 24.73, 24.60, 23.10, 22.77, 22.70, 22.58, 22.48, 22.37, 21.68, 20.94, 20.13, 17.26, 15.97, 14.57, 14.32, 13.11; ESI-MS m/z 756.5 [M + H]+.Yellow solid , yield 30.40%. 1H NMR  δ 8.69 , 8.38 , 7.40 , 7.32 − 7.31 , 5.34 , 3.78 , 3.52 , 3.27 − 3.16 , 2.94 , 2.52 − 2.45 , 2.35 − 2.24 , 2.23 − 2.15 , 2.10 , 2.05 − 1.98 , 1.95 − 1.88 , 1.88 − 1.86 , 1.76 , 1.63 − 1.58 , 1.57 − 1.53 , 1.43 , 1.40 − 1.31 , 1.30 − 1.23 , 1.22 , 1.17 − 1.13 , 1.00 , 0.98 , 0.96 , 0.89 , 0.85 , 0.78 ; 13C NMR  δ 177.39, 173.35, 151.55(C5-Tacrine), 144.63, 142.13, 140.84, 135.01, 129.33(C6-Tacrine), 129.23, 128.29, 123.72(C7-Tacrine), 122.60, 120.63(C8-Tacrine), 55.25, 55.09, 47.62, 47.52, 47.50, 46.75, 46.30, 45.60, 42.28, 42.17, 41.92, 41.64, 39.48, 39.37, 38.76, 38.73, 38.60, 38.52, 38.47, 37.01, 36.96, 34.13, 33.70, 32.96, 32.94, 32.78, 32.53, 32.16, 30.69, 30.62, 28.11, 28.09, 27.31, 27.18, 27.15, 25.72, 25.59, 23.60, 23.52, 23.50, 23.46, 23.31, 18.35, 18.25, 17.56, 16.92, 15.58, 15.56, 15.38; ESI-MS m/z 770.5 [M + H]+.Yellow solid , yield 22.06%. 1H NMR  δ 8.29 , 8.08 , 7.30 , 5.43 , 4.15 , 3.84 , 3.66 , 3.23 − 3.09 , 2.72 − 2.57 , 2.03 , 1.91 − 1.86 , 1.77 − 1.68 , 1.64 − 1.57 , 1.57 − 1.51 , 1.29 − 1.23 , 1.15 , 0.97 , 0.91 , 0.83 , 0.75 , 0.70 , 0.64 ; 13C NMR  δ 180.97, 143.22(C6-Tacrine), 126.80(C5-Tacrine), 125.38(C7-Tacrine), 122.34(C8-Tacrine), 54.04, 52.42, 50.64, 46.43, 45.55, 45.35, 40.94, 40.79, 38.95, 38.34, 37.73, 37.40, 35.93, 33.04, 31.95, 31.78, 31.22, 29.72, 28.77, 28.69, 28.35, 28.31, 27.63, 27.05, 26.28, 26.20, 26.11, 24.76, 22.97, 22.66, 22.60, 22.47, 21.68, 21.05, 19.78, 17.22, 15.76, 14.54, 14.34, 14.30, 13.12; ESI-MS m/z 758.5 [M + H]+.Brown solid , yield 19.78%. 1H NMR  δ 12.57 , 7.57 , 7.51 , 7.14 , 7.09 , 4.41 , 3.59 , 3.32 , 3.25 , 2.93 , 2.73 , 2.45 , 2.32 , 2.25 , 1.21 , 1.14 , 1.06 − 0.98 , 0.91 , 0.85 − 0.79 , 0.70 , 0.68 − 0.64 , 0.62 , 0.60 , 0.58 , 0.57 , 0.55 , 0.54 , 0.38 − 0.35 , 0.31 , 0.26 , 0.17 , 0.16 , 0.13 , 0.08 , −0.06 , −0.13 ; 13C NMR  δ 181.33, 159.18, 114.35, 92.19, 91.24, 76.16, 75.96, 75.88, 75.52, 74.04, 71.09, 70.42, 70.30, 69.60, 68.00, 65.80, 64.52, 64.42, 63.12, 61.03, 60.40, 59.86, 58.94, 58.16, 55.72, 55.40, 54.37, 53.83, 53.54, 52.35, 50.13; ESI-MS m/z 772.4 [M + H]+.Brown solid , yield 23.30%. 1H NMR  δ 8.18 , 7.65 , 7.21 , 6.83 , 5.45 , 4.12 − 3.99 , 3.79 , 3.63 − 3.56 , 3.19 , 3.01 , 2.65 , 2.04 , 1.93 , 1.90 − 1.86 , 1.74 , 1.70 − 1.62 , 1.61 − 1.55 , 1.51 , 1.45 − 1.39 , 1.39 − 1.32 , 1.27 − 1.22 , 1.22 − 1.17 , 1.14 , 1.04 , 0.95 , 0.90 , 0.81 , 0.73 , 0.68 , 0.60 ; 13C NMR  δ 181.29, 181.27, 164.75, 154.97, 154.94, 149.85, 142.58(C6-Tacrine), 142.56, 138.50, 137.53, 137.49, 126.19(C5-Tacrine), 124.39, 124.34(C7-Tacrine), 122.67(C8-Tacrine), 117.81, 113.10, 111.32, 111.28, 54.04, 50.79, 46.44, 45.48, 45.32, 40.85, 40.60, 40.58, 38.65, 38.32, 37.70, 37.66, 37.39, 35.89, 33.06, 31.96, 31.86, 31.26, 29.67, 28.68, 27.72, 27.05, 26.27, 26.10, 24.75, 22.66, 22.52, 22.48, 22.41, 20.92, 19.84, 17.21, 15.64, 14.54, 14.20; ESI-MS m/z 786.4 [M + H]+.Brown solid , yield 27.98%. 1H NMR  δ 8.02 , 7.94 , 7.44 , 6.17 , 5.36 , 3.78 , 3.36 , 3.19 , 3.09 − 3.02 , 2.98 , 2.60 , 2.57 − 2.50 , 2.00 − 1.94 , 1.90 , 1.86 , 1.82 , 1.73 , 1.68 − 1.60 , 1.57 , 1.55 , 1.50 , 1.46 − 1.39 , 1.36 − 1.29 , 1.23 , 1.13 , 1.02 , 0.96 , 0.88 , 0.83 , 0.74 , 0.69 ; 13C NMR  δ 177.39, 173.35, 151.55(C6-Tacrine), 142.13, 140.84, 135.01, 129.33(C5-Tacrine), 129.23, 128.29, 123.72(C7-Tacrine), 122.60, 120.63(C8-Tacrine), 55.25, 55.09, 47.62, 47.52, 47.50, 46.75, 46.30, 45.60, 42.28, 42.17, 41.93, 41.64, 39.48, 39.37, 38.77, 38.76, 38.73, 38.60, 38.53, 38.47, 37.01, 36.96, 34.13, 33.70, 32.96, 32.94, 32.78, 32.53, 32.17, 30.69, 30.62, 28.11, 28.09, 27.31, 27.18, 27.15, 25.72, 25.59, 23.60, 23.52, 23.50, 23.46, 23.31, 18.35, 18.25, 17.56, 16.92, 15.58, 15.56, 15.39; ESI-MS m/z 800.5 [M + H]+; HRMS(ESI) m/z [M + H]+ calcd for C48H71N3BrO2+ 800.4730, found 800.4744.Brown solid , yield 41.23%. 1H NMR  δ 8.07 , 8.05 , 7.50 , 6.01 , 5.37 , 3.85 , 3.35 − 3.29 , 3.21 , 3.04 , 3.02 − 2.95 , 2.61 , 2.50 , 1.99 − 1.93 , 1.91 , 1.81 , 1.75 − 1.67 , 1.60 , 1.57 − 1.52 , 1.46 , 1.43 − 1.38 , 1.38 − 1.33 , 1.31 , 1.16 , 1.06 1.02 , 0.98 , 0.90 , 0.77 , 0.72 ; 13C NMR  δ 177.40, 154.33, 151.25, 143.96(C6-Tacrine), 139.48, 127.40(C5-Tacrine), 125.86, 125.29(C7-Tacrine), 122.38, 121.81(C8-Tacrine), 113.82, 111.32, 54.06, 52.42, 49.83, 47.87, 46.50, 45.75, 45.26, 41.35, 41.10, 38.55, 38.36, 37.75, 37.46, 35.95, 33.13, 31.96, 31.57, 31.37, 29.72, 28.69, 28.64, 28.51, 28.47, 28.41, 28.35, 28.33, 28.31, 28.24, 28.14, 28.11, 28.03, 27.09, 26.28, 26.11, 26.04, 25.55, 24.71, 22.83, 22.56, 22.53, 22.28, 20.83, 19.88, 17.27, 15.96, 14.59, 14.37, 13.11; ESI-MS m/z 814.4 [M + H]+.Brown solid , yield 25.79%. 1H NMR  δ 8.23 , 8.02 , 7.24 , 7.13 , 5.37 , 4.08 , 3.78 , 3.59 , 3.15 − 3.09 , 2.63 − 2.57 , 1.88 − 1.76 , 1.56 − 1.44 , 1.42 − 1.34 , 1.33 − 1.25 , 1.21 − 1.17 , 1.15 , 1.09 , 0.99 , 0.90 , 0.84 , 0.77 , 0.69 , 0.63 , 0.58 ; 13C NMR  δ 181.92, 155.49, 144.16(C5-Tacrine), 127.74(C6-Tacrine), 126.32(C7-Tacrine), 123.27(C8- Tacrine), 114.25, 111.82, 54.97, 53.36, 51.57, 50.80, 47.37, 46.48, 46.28, 41.87, 41.72, 39.89, 39.27, 39.15, 38.66, 38.33, 36.86, 33.98, 32.89, 32.71, 32.15, 31.85, 30.66, 29.70, 29.63, 29.29, 29.25, 29.18, 28.56, 27.99, 27.22, 27.14, 27.04, 25.69, 23.90, 23.59, 23.53, 23.48, 23.40, 22.62, 21.98, 20.71, 18.16, 16.70, 15.48, 15.24, 14.05; ESI-MS m/z 758.4 [M + H]+.Brown solid , yield 21.10%. 1H NMR  δ 10.08 , 8.30 , 8.01 , 7.46 , 5.47 , 3.64 , 3.23 , 3.11 , 2.78 − 2.71 , 2.65 − 2.59 , 2.08 − 2.03 , 1.96 , 1.90 , 1.74 , 1.70 − 1.64 , 1.61 − 1.50 , 1.22 , 1.15 , 1.07 , 0.97 , 0.90 , 0.86 , 0.76 , 0.71 ; 13C NMR  δ 177.62, 166.77, 164.96, 156.32, 149.47, 143.50(C5-Tacrine), 135.58, 134.22, 133.22, 131.42, 129.89, 128.90(C6-Tacrine), 128.87, 128.47(C7-Tacrine), 127.78(C8-Tacrine), 124.84, 124.57, 121.96, 115.66, 111.95, 111.74, 77.90, 76.23, 76.02, 75.81, 67.14, 66.75, 54.40, 54.06, 47.97, 46.51, 45.68, 45.23, 40.97, 38.33, 37.98, 37.88, 37.73, 37.71, 37.41, 35.93, 33.15, 31.99, 31.71, 31.37, 30.91, 29.69, 29.55, 29.34, 29.29, 28.68, 28.67, 28.64, 28.60, 28.35, 28.31, 28.08, 28.03, 27.97, 27.91, 27.08, 26.27, 26.20, 26.13, 24.75, 22.96, 22.95, 22.72, 22.69, 22.59, 22.46, 21.97, 21.95, 21.68, 20.52, 19.51, 17.54, 17.26, 16.11, 15.97, 14.57, 14.32, 13.11, 13.04, 10.08, 9.95; ESI-MS m/z 772.4 [M + H]+.Brown solid , yield 23.31%. 1H NMR  δ 9.92 , 8.36 , 8.06 , 7.50 , 6.42 , 5.48 , 4.05 , 3.78 − 3.71 , 3.44 , 3.26 , 3.14 , 3.01 , 2.76 , 2.68 − 2.63 , 2.00 , 1.97 − 1.92 , 1.79 , 1.67 , 1.51 − 1.48 , 1.46 , 1.31 , 1.20 , 1.10 , 1.03 , 0.95 , 0.91 , 0.82 , 0.78 ; 13C NMR  δ 178.61, 167.70, 165.90, 157.30, 144.55(C5-Tacrine), 136.48, 135.17, 134.17, 132.37(C6-Tacrine), 130.82, 129.42, 128.73(C7-Tacrine), 125.69(C8-Tacrine), 125.50, 122.94, 116.68, 112.97, 112.79, 78.86, 77.16, 76.95, 76.74, 68.09, 67.70, 55.01, 48.30, 47.46, 46.65, 46.19, 42.01, 41.96, 39.28, 38.83, 38.81, 38.68, 38.65, 38.37, 36.89, 34.08, 32.91, 32.57, 32.30, 31.86, 30.64, 30.50, 30.28, 29.97, 29.63, 29.25, 29.12, 29.10, 28.91, 28.85, 28.02, 27.20, 27.08, 25.73, 25.69, 25.40, 23.91, 23.70, 23.67, 23.53, 23.45, 22.92, 22.90, 22.62, 21.54, 20.50, 18.21, 16.90, 15.51, 15.30, 14.06, 13.99, 11.03, 10.89; ESI-MS m/z 786.4 [M + H]+.Brown solid , yield 31.79%. 1H NMR  δ 9.94 , 8.37 , 8.07 , 7.59 , 7.50 , 5.46 , 4.01 , 3.42 , 3.26 , 3.14 , 3.09 , 2.75 , 2.65 − 2.58 , 2.01 , 1.98 − 1.93 , 1.83 − 1.77 , 1.76 − 1.70 , 1.68 , 1.63 − 1.59 , 1.42 − 1.34 , 1.31 , 1.21 , 1.12 − 1.05 , 1.04 , 0.95 , 0.93 , 0.82 , 0.80 ; 13C NMR  δ 177.60, 166.75, 164.94, 156.30, 149.45, 143.48(C5-Tacrine), 135.56, 134.20, 133.20(C6-Tacrine), 131.40, 129.87, 128.88, 128.85, 128.45, 127.76, 124.82(C7-Tacrine), 124.55, 121.94(C8-Tacrine), 115.64, 111.93, 111.72, 77.88, 76.21, 76.00, 75.79, 67.12, 66.73, 54.38, 54.04, 47.95, 46.49, 45.66, 45.21, 42.41, 40.95, 38.31, 37.96, 37.86, 37.71, 37.69, 37.39, 35.91, 33.13, 31.97, 31.69, 31.35, 30.89, 29.67, 29.53, 29.32, 29.27, 28.66, 28.65, 28.62, 28.58, 28.33, 28.29, 28.06, 28.01, 27.95, 27.89, 27.06, 26.25, 26.18, 26.11, 24.73, 22.94, 22.93, 22.70, 22.67, 22.57, 22.44, 21.95, 21.93, 21.66, 20.50, 19.49, 17.52, 17.24, 16.09, 15.95, 14.55, 14.30, 13.09, 13.02, 11.57, 10.06, 9.93; ESI-MS m/z 800.4 [M + H]+.Brown solid , yield 38.73%. 1H NMR  δ 10.70 , 8.29 , 7.76 − 7.69 , 7.48 , 6.76 , 5.42 , 4.17 − 4.06 , 3.71 , 3.19 , 3.01 , 2.73 − 2.67 , 2.63 , 1.96 − 1.81 , 1.13 , 0.95 , 0.89 , 0.75 , 0.52 ; 13C NMR  δ 182.30, 165.72, 157.19, 143.71(C5-Tacrine), 133.12(C6-Tacrine), 125.36, 123.56(C7-Tacrine), 119.50(C8-Tacrine), 115.69, 111.52, 56.01, 55.90, 55.78, 55.02, 51.89, 47.40, 46.52, 46.35, 43.82, 41.87, 41.64, 39.74, 39.28, 38.72, 38.66, 38.39, 36.88, 34.02, 32.97, 32.72, 32.24, 30.68, 29.71, 28.31, 28.03, 27.25, 27.07, 25.74, 23.53, 23.50, 23.41, 21.98, 20.78, 18.52, 18.20, 18.15, 17.03, 16.67, 16.61, 15.50, 15.18, 12.75; ESI-MS m/z 814.4 [M + H]+.Brown solid , yield 33.15%. 1H NMR  δ 13.37 , 8.54 , 7.90 , 7.79 , 7.49 , 5.12 , 4.28 , 4.02 − 3.91 , 3.80 − 3.72 , 3.62 , 3.57 − 3.51 , 3.42 , 3.14 , 3.07 − 3.01 , 2.97 − 2.92 , 2.66 , 1.84 , 1.75 − 1.69 , 1.62 − 1.49 , 1.39 , 1.32 , 1.01 , 0.87 , 0.85 , 0.83 , 0.63 , 0.57 , 0.13 ; 13C NMR  δ 178.90, 164.89, 163.22, 162.77, 162.01, 160.59, 156.46, 144.17(C6-Tacrine), 130.11(C5-Tacrine), 122.01(C7-Tacrine), 111.70(C8-Tacrine), 77.18, 55.39, 47.28, 46.29, 45.74, 42.31, 41.53, 36.88, 36.25, 33.92, 33.83, 33.25, 33.12, 32.46, 31.23, 30.84, 29.56, 29.51, 28.64, 28.42, 27.35, 27.25, 25.95, 24.71, 23.88, 23.24, 22.68, 22.34, 21.83, 20.93, 18.55, 18.30, 17.20, 16.67, 16.41, 15.19, 12.96; ESI-MS m/z 698.5 [M + H]+.Brown solid , yield 24.34%. 1H NMR  δ 13.38 , 8.49 , 7.52 − 7.48 , 7.48 − 7.45 , 7.41 , 5.11 , 4.29 , 3.83 , 3.19 , 3.08 , 2.95 , 2.65 , 1.91 − 1.85 , 1.85 − 1.80 , 1.74 − 1.67 , 1.64 − 1.57 , 1.50 , 1.40 , 1.39 − 1.36 , 1.35 , 1.32 , 1.30 − 1.20 , 1.04 , 1.01 , 0.88 , 0.83 , 0.77 , 0.65 , 0.59 , 0.41 ; 13C NMR  δ 177.17, 165.06, 164.87, 163.20, 162.77, 156.32, 144.45(C6-Tacrine), 121.75(C5-Tacrine), 114.89(C7-Tacrine), 111.94(C8-Tacrine), 77.20, 55.08, 47.37, 46.37, 46.33, 45.69, 41.61, 38.41, 36.91, 36.43, 36.25, 34.03, 33.33, 33.30, 32.69, 31.24, 30.85, 30.41, 28.67, 27.38, 27.33, 26.01, 23.95, 23.23, 22.71, 21.86, 20.83, 18.36, 17.09, 16.44, 15.33; ESI-MS m/z 712.5 [M + H]+.Brown solid , yield 36.49%.1H NMR  δ 13.34 , 8.44 , 7.48 , 7.23 , 5.10 , 4.30 , 3.80 , 3.12 − 3.06 , 2.95 , 2.72 , 2.64 , 1.83 , 1.66 , 1.61 , 1.45 , 1.42 − 1.37 , 1.34 , 1.23 , 1.03 , 0.98 , 0.87 , 0.85 , 0.83 , 0.64 , 0.60 , 0.48 ; 13C NMR  δ 176.72, 144.55, 121.70, 77.19, 55.10, 47.41, 46.45, 38.87, 38.80, 38.42, 36.91, 34.07, 33.37, 33.21, 32.84, 30.87, 29.49, 28.68, 27.97, 27.38, 26.88, 26.03, 23.97, 23.27, 22.71, 22.00, 18.35, 17.26, 16.47, 15.33; ESI-MS m/z 726.5 [M + H]+.Brown solid , yield 30.28%.1H NMR  δ 13.43 , 8.47 , 7.54 − 7.50 , 7.50 − 7.45 , 7.21 , 5.15 , 4.29 , 3.80 , 3.04 , 2.98 , 2.96 − 2.94 , 2.76 , 2.73 , 2.63 , 2.02 − 1.95 , 1.88 , 1.83 , 1.75 − 1.69 , 1.63 , 1.56 − 1.49 , 1.40 , 1.30 , 1.24 − 1.21 , 1.15 − 1.11 , 1.05 , 1.02 − 0.99 , 0.87 , 0.85 , 0.78 , 0.69 , 0.67 , 0.62 , 0.57 ; 13C NMR  δ 176.62, 164.74, 163.06, 162.78, 155.86, 144.60(C6-Tacrine), 130.11(C5-Tacrine), 121.73(C7-Tacrine), 114.92(C8-Tacrine), 114.76, 113.30, 111.92, 77.21, 70.26, 55.39, 55.15, 48.07, 47.46, 46.49, 45.67, 38.98, 38.79, 38.72, 38.44, 36.94, 36.25, 34.09, 33.38, 33.22, 32.86, 31.76, 31.24, 30.89, 29.85, 29.56, 29.50, 29.26, 29.17, 29.05, 28.76, 28.67, 27.38, 26.06, 24.19, 24.01, 23.31, 22.75, 22.57, 21.89, 20.85, 18.37, 17.31, 16.44, 15.39, 14.43; ESI-MS m/z 740.5 [M + H]+.Brown solid , yield 44.57%.1H NMR  δ 13.38 , 8.45 , 7.51 , 7.50 , 7.20 , 5.16 , 4.29 , 3.81 , 3.06 − 3.01 , 2.95 , 2.76 , 2.63 , 1.83 , 1.71 , 1.64 , 1.54 , 1.44 − 1.41 , 1.41 − 1.35 , 1.35 − 1.31 , 1.27 − 1.23 , 1.19 − 1.14 , 1.13 − 1.09 , 1.05 , 1.02 − 1.00 , 0.86 − 0.84 , 0.71 , 0.63 , 0.60 , 0.59 ; 13C NMR  δ 176.57, 164.80, 163.12, 155.97, 144.64(C6-Tacrine), 121.71(C5-Tacrine), 115.00(C7-Tacrine), 114.84(C8-Tacrine), 111.76, 77.19, 55.39, 55.15, 47.85, 47.48, 46.49, 45.67, 41.70, 40.92, 39.06, 38.77, 38.45, 36.95, 34.09, 33.40, 33.24, 32.88, 30.89, 30.20, 29.41, 28.64, 27.39, 26.61, 26.27, 26.08, 24.02, 23.34, 22.73, 21.86, 20.80, 18.37, 17.36, 16.41, 15.39; ESI-MS m/z 754.5 [M + H]+.Brown solid , yield 31.81%.1H NMR  δ 7.99 , 7.95 , 7.43 − 7.38 , 7.29 , 5.05 , 4.27 , 3.68 − 3.55 , 3.39 , 3.25 , 2.97 − 2.94 , 2.91 , 2.89 , 2.73 , 1.88 , 1.81 , 1.70 , 1.62 − 1.55 , 1.48 , 1.40 , 1.37 − 1.32 , 1.24 − 1.20 , 1.12 − 1.03 , 1.00 , 0.86 , 0.83 , 0.79 − 0.73 , 0.65 , 0.60 − 0.54 , 0.29 ; 13C NMR  δ 177.84, 162.76, 144.29(C5-Tacrine), 123.00(C6-Tacrine), 121.93(C7-Tacrine), 120.07(C8-Tacrine), 77.25, 55.38, 55.14, 49.03, 47.41, 46.41, 45.67, 41.55, 40.84, 38.80, 38.71, 36.92, 36.24, 33.99, 33.29, 33.07, 32.47, 31.23, 30.83, 28.66, 27.38, 27.30, 26.00, 25.33, 23.93, 23.25, 22.88, 22.86, 22.49, 18.30, 16.81, 16.41, 15.31; ESI-MS m/z 698.5 [M + H]+.Black solid , yield 22.91%. 1H NMR  δ 8.02 , 7.95 , 7.49 , 7.37 − 7.32 , 5.11 , 4.27 , 3.59 − 3.50 , 3.17 , 3.04 − 2.99 , 2.95 , 1.81 , 1.72 , 1.62 , 1.51 , 1.43 − 1.39 , 1.36 , 1.28 , 1.26 − 1.22 , 1.07 , 1.01 , 0.91 , 0.87 , 0.84 , 0.79 , 0.67 , 0.61 − 0.57 , 0.43 ; 13C NMR  δ 177.07, 165.06, 162.77, 144.46(C6-Tacrine), 123.59(C5-Tacrine), 121.77(C5-Tacrine), 120.21(C8-Tacrine), 77.25, 55.17, 47.43, 46.44, 46.29, 45.68, 41.63, 36.93, 36.68, 36.25, 34.07, 33.34, 33.30, 32.74, 31.23, 31.02, 30.85, 29.52, 28.68, 27.40, 27.35, 26.02, 25.36, 23.97, 23.27, 22.73, 22.69, 22.57, 22.18, 18.35, 17.10, 16.43, 15.36; ESI-MS m/z 712.5 [M + H]+.Black solid , yield 28.07%. 1H NMR  δ 13.24 , 8.19 , 7.82 , 7.56 , 7.24 , 5.11 , 4.31 − 4.27 , 3.84 , 3.11 , 3.02 , 2.96 , 2.75 − 2.70 , 2.67 , 2.02 − 1.95 , 1.69 , 1.61 , 1.52 , 1.46 , 1.43 − 1.36 , 1.25 , 1.23 , 1.02 , 0.88 − 0.81 , 0.62 , 0.61 − 0.56 , 0.46 ; 13C NMR  δ 176.73, 155.80, 144.54(C6-Tacrine), 130.12(C5-Tacrine), 125.16(C7-Tacrine), 121.69, 112.61(C8-Tacrine), 77.18, 55.39, 55.08, 47.59, 47.40, 46.45, 45.67, 41.68, 39.22, 38.87, 38.78, 38.42, 36.89, 34.07, 33.37, 33.22, 32.82, 30.87, 29.56, 29.50, 29.17, 28.66, 28.05, 27.37, 26.88, 26.01, 23.97, 23.26, 22.71, 22.57, 21.84, 20.77, 18.29, 17.25, 16.45, 15.30, 14.43; ESI-MS m/z 726.5 [M + H]+.Black solid , yield 38.54%. 1H NMR  δ 8.23 , 7.53 , 7.17 , 6.12 , 5.37 , 3.82 , 3.38 , 3.21 , 3.06 , 3.02 , 2.61 , 2.53 , 1.99 , 1.91 − 1.87 , 1.75 , 1.69 − 1.62 , 1.56 , 1.48 − 1.44 , 1.37 − 1.31 , 1.22 − 1.17 , 1.15 , 1.04 , 0.98 , 0.89 , 0.86 , 0.76 , 0.75 , 0.73 − 0.69 ; 13C NMR  δ 178.96, 165.00, 163.30, 155.34, 152.82, 144.82(C6-Tacrine), 141.99, 141.90, 127.93(C5-Tacrine), 127.86, 123. 08(C7-Tacrine), 115.05, 114.89, 113.46, 112.10(C8-Tacrine), 106.24, 106.08, 55.20, 49.02, 47.63, 46.86, 46.45, 42.27, 42.19, 39.50, 39.13, 38.88, 38.57, 37.08, 34.27, 33.10, 32.88, 32.50, 32.06, 30.84, 30.30, 29.84, 29.79, 29.66, 29.53, 29.50, 29.48, 29.46, 29.38, 28.23, 27.42, 27.26, 25.88, 24.21, 23.96, 23.70, 23.63, 23.46, 22.83, 22.06, 21.06, 18.40, 17.13, 15.71, 15.45, 14.26; ESI-MS m/z 740.5 [M + H]+.Black solid , yield 47.27%. 1H NMR  δ 8.08 , 7.60 , 7.48 − 7.41 , 7.19 , 5.16 , 4.28 , 3.65 , 3.02 , 2.99 − 2.96 , 2.95 − 2.92 , 2.75 , 2.68 , 1.82 , 1.76 − 1.68 , 1.66 − 1.62 , 1.56 − 1.50 , 1.40 , 1.34 , 1.29 , 1.25 − 1.21 , 1.14 − 1.09 , 1.05 , 0.85 , 0.71 , 0.60 ; 13C NMR  δ 176.54, 162.78, 144.64(C6-Tacrine), 124.21(C5-Tacrine), 121.73(C7-Tacrine), 121.21(C8-Tacrine), 77.21, 55.17, 48.05, 47.50, 46.50, 45.66, 41.69, 38.77, 38.46, 36.96, 34.10, 33.40, 33.23, 32.88, 30.90, 30.63, 29.47, 28.65, 27.39, 26.72, 26.42, 26.08, 24.96, 24.03, 23.34, 22.73, 22.30, 21.59, 18.36, 17.34, 16.40, 15.39; ESI-MS m/z 754.5 [M + H]+.Black solid , yield 25.18%. hAChE, hBuChE, Acetylcholinesterase Activity Assay Kit and Butyrylcholinesterase Activity Assay Kit were purchased from Sigma Aldrich. And tacrine hydrochloride were purchased from MedChemExpress. The capacity of all the target compounds to inhibit hAChE and hBuChE activities were assessed by Ellman’s method. The concentration of compound producing 50% of enzyme activity inhibition (IC50) was calculated by nonlinear regression analysis of the response-concentration (log) curve, using the Graph-Pad Prism program package . Results are expressed as the mean ± SD of at least three different experiments performed in triplicate.Preparation of 50 mM Tris–HCl buffer solutions: 5 ml Tris solution  was dissolved in distilled water (95 ml) and adjusted with HCl to a pH of 8.0 ± 0.1. Buffer was freshly prepared and stored in the refrigerator. AChE solution 2.005 U/ml: the enzyme  was dissolved in freshly prepared buffer pH 8.0 (5 ml). BChE solution 2.040 U/ml: the enzyme  was dissolved in freshly prepared buffer pH 8.0 (5 ml). DTNB solution 3 mM: DTNB (23.8 mg) was dissolved in freshly prepared buffer pH 8.0 (20 ml) containing NaCl (116.8 mg) and MgCl2 (38.0 mg). ATChI solution 15 mM: ATChI (43.4 mg) was dissolved in distilled water (10 ml). BTChI solution 15 mM: BTChI (47.6 mg) was dissolved in distilled water (10 ml). All solutions were stored in Eppendorf caps in the refrigerator or freezer, if necessary. The pure compounds were initially dissolved in DMSO, Tacrine as standard was dissolved in distilled water. The final concentrations for the enzymatic assay were yielded by diluting the stock solution with bi-distilled water. No inhibition was detected by residual DMSO (<0.5%).A mixture of the DTNB solution (160 µL), enzyme solution (50 µL) and compounds solutions  was prepared and incubated at 37 °C for 10 min. The substrate (30 µL) was added to start the enzymatic reaction. The absorbance data (l = 405 nm) was recorded under a controlled temperature of 30 °C for 15 min at 1 min intervals. All measurements were performed as triplicates.Molecular docking studies were performed using the Discovery Studio 2.0 software program (DS 2.0). The X-ray crystallographic structure of AChE (PDB code 2CKM) was obtained from the PDB (the Protein Data Bank). First, Water molecules were removed. Second, hydrogen atoms were added. Third, side chain amides and side chains bumps were fixed. The compounds B4 and D4 was imported to DS 2.0 and docked into the active site to investigate the binding modes.−1 penicillin/streptomycin and maintained in a humidified atmosphere of 5% CO2 at 37 °C. Initially, 8000 cells per well were seeded in 96-well plates for HepG2 or SH-SY5Y cells, then treated with vehicle alone or tested compounds for 24 h. Then 10 μL CCK8 purchased from CELLCOOK was added to each well and further incubated for another 3 h. The absorbance was measured using a microplate reader (450 nm).HepG2 and SH-SY5Y cells were obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences . HepG2 and SH-SY5Y cells were cultured in MEM or MEM/F12 supplemented with 10% FBS, 100 units mLμL of binding buffer. After stained with 5 μL AnnexinV-FITC and 5 μL propidium iodide at room temperature for 15 min. Cells were then analysed by BD Accuri C6 flow cytometer with cell quest software . Cells undergoing apoptosis are both Annexin V positive and PI negative.Annexin V-FITC and propidium iodide were used to evaluate apoptotic cells by flow cytometry. Cells were treated with different concentrations of tested compounds for 24 h. Then the cells were washed twice with phosphate-buffered saline (PBS) . The collected cells were then resuspended in 500 5 cells/well, treating with different tested compounds for 24 h, and then washed three times and incubated with final concentration of 10 μM DCFH-DA for 30 min at 37 °C in the dark. After incubation, cells were washed three times and harvested in free-serum medium. The fluorescence of 2′, 7′-dichlorofluorescein (DCF) was detected by flow cytometry (488 nm excitation and 525 nm emission filters) using BD Accuri C6 flow cytometer . Data were processed by using cell quest software .The level of intracellular ROS was measured by using the ROS-sensitive dye, 2′,7′-dichloro-fluorescein diacetate (DCFH-DA), as a probe. In brief, cells were seeded in six-well plates at 2.0 × 10


Aedes aegypti is a vector of several arboviruses, notably dengue virus (DENV), which causes dengue fever and is often found resting indoors. Culex spp. are largely nuisance mosquitoes but can include species that are vectors of zoonotic pathogens. Vector control is currently the main method to control dengue outbreaks. Indoor residual spraying can be part of an effective vector control strategy but requires an understanding of the resting behavior. Here we focus on the indoor-resting behavior of Ae. aegypti and Culex spp. in northeastern Thailand.Ae. aegypti, Aedes albopictus and Culex spp. Dengue virus was detected in Ae. aegypti. Association analyses between urban/rural and within-house location , household variables, geckos and mosquito abundance were performed.Mosquitoes were collected in 240 houses in rural and urban settings from May to August 2019 at two collection times (morning/afternoon), in four room types  in each house and at three wall heights  using a battery-driven aspirator and sticky traps. Household characteristics were ascertained. Mosquitoes were identified as Aedes aegypti and Culex spp. accounted for 44.78% and 53.17% of the specimens, respectively. Only 2.05% were Ae. albopictus. Aedes aegypti and Culex spp. rested most abundantly at intermediate and low heights in bedrooms or bathrooms . Clothes hanging at intermediate heights were associated with higher mean numbers of Ae. aegypti in rural settings (0.81 [SEM: 0.08] vs. low: 0.61 [0.08] and high: 0.32 [0.09]). Use of larval control was associated with lower numbers of Ae. aegypti . All DENV-positive Ae. aegypti  were collected in the rural areas and included specimens with single, double and even triple serotype infections.A total of 2874 mosquitoes were collected using aspirators and 1830 using sticky traps. Knowledge of the indoor resting behavior of adult mosquitoes and associated environmental factors can guide the choice of the most appropriate and effective vector control method. Our work suggests that vector control using targeted indoor residual spraying and/or potentially spatial repellents focusing on walls at heights lower than 1.5 m in bedrooms and bathrooms could be part of an integrated effective strategy for dengue vector control.The online version contains supplementary material available at 10.1186/s13071-023-05746-9. Aedes aegypti  is a tropical and subtropical mosquito species widely distributed globally. It is a primary vector of the dengue virus (DENV) and is well adapted to completing its entire life cycle within urban areas and around houses, primarily feeding on humans. It also transmits yellow fever, Zika and chikungunya viruses. Aedes albopictus is a secondary vector of DENV and, although more rural, also exhibits peridomestic resting and biting behaviors.Dengue is the most widespread mosquito-borne viral disease in the world. The number of dengue cases reported to the World Health Organization has increased more than eight-fold over the last two decades . An estiAe. aegypti generally rest indoors rather than outdoors  0.13 vs. sticky trap mean: 3.98, SEM: 0.22. Culex spp. aspirator mean: 0.91, SEM: 0.19 vs. sticky trap mean: 3.86, SEM: 0.19). However, per sampling sticky traps were deployed for a lot longer . For the comparison, only aspirator mosquito collections from houses that had sticky traps deployed were used. Taking into account the differences between area, village, room and height, the aspirator method was more efficient per sampling time effort in collecting Ae. aegypti  and Culex spp.  mosquitoes. Aspirators collected a mean of 0.019 Ae. aegypti/min and 0.029 Culex spp./min, whereas sticky traps collected a mean of 1.3 × 10–4Ae. aegypti/min and 1.28 × 10–4Culex spp./min. Although the efficiency of capture was higher at all heights, for Ae. aegypti there was a significant interaction effect where aspiration was even better at higher heights .Overall, in houses where mosquitoes were collected both by aspiration and sticky traps, the sticky traps collected more mosquitoes  were positive for the virus. This was too few to analyze with respect to their distribution in rooms and across heights. Two specimens were positive for DENV-1 (0.7%), one for DENV-3 (0.3%), and one with a mixed DENV-1 and DENV-3 infection (0.3%). One specimen was positive for DENV-1, DENV-2, and DENV-3 combined (0.3%). All DENV-positive mosquitoes were collected by aspiration in May 2019 in the rural village of Ku Thong, Mahasarakham province. These five specimens were found in one positive pool of 10 mosquitoes. Three positive mosquitoes were found in the living rooms of two houses, and two mosquitoes were found in the bedroom of one house. All five positive mosquitoes were found at a height of 0.75–1.5 m.Of the 422 Summary household information collected using the questionnaire is shown in Additional file P-values for the univariable analyses of the association of socioenvironmental variables with Ae. aegypti or Culex spp. mosquito numbers are shown in Additional file Ae. aegypti number, and the Yes category was found to be associated with increased numbers of mosquitoes . In rural settings, the following variables were found to be associated with higher Ae. aegypti number: use of temephos for larval control with low use (only every 3 months) was associated with higher numbers of mosquitoes ; cement walls , outdoor toilets  and increasing number of rooms . Clothing location was also associated with mosquito numbers , with intermediate height of hung clothing being associated with more mosquitoes than lower or higher levels . With 38 variables analyzed and one multivariable analysis would yield a Bonferroni-corrected P-value of P = 0.0013. Thus, the associations observed above must be taken with caution.The Culex spp. mosquitoes in urban settings, the following variables were found to be associated with higher mosquito numbers: absence of open eaves , infrequent use of fogging (every 3 months)  and low levels of wind flow . In rural settings, the use of fogging  was associated with lower numbers of mosquitoes, and the quantity of hung clothing  was associated with higher numbers of mosquitoes. Shopkeeping as the household occupation was also associated with higher mosquito numbers . As above, the application of the Bonferroni-corrected P-value threshold would suggest that only the use of fogging can be safely considered associated with lower mosquito numbers.For N = 82), followed by bedrooms , living rooms  and kitchens . Geckos were caught on the lower , intermediate  and upper levels .A total of 297 geckos were caught, of which 133 (45%) were in the urban area and 164 (55%) in the rural area. Close to 28% of the geckos were collected in bathrooms . By contrast, there was a positive association between gecko number and the total number of Culex spp. caught by aspiration . These associations remained in a multivariable analysis that included the above-identified significant socioenvironmental variables.Univariable fitting of the gecko number caught revealed no significant association of gecko number with either the Ae. aegypti mosquito resting behavior and environmental factors therewith associated. We found that the majority of female Ae. aegypti mosquitoes rested in bedrooms (35–39% of specimens) and bathrooms (ca. 30%) and at intermediate heights (48–53% of specimens). Similar results were found in Trinidad, Mexico and Panama, where female Ae. aegypti generally rested in bedrooms, but less so in bathrooms [Culex spp. were also predominantly found in bedrooms and bathrooms and at low and intermediate heights. The similarities in place and height of resting across the genera are interesting but must be treated with caution, especially as the Culex spp. were not identified to the species level. Mosquitoes are abundant in bedrooms likely because people spend a comparatively long time in this room while sleeping, attracting blood-seeking mosquitoes by their body heat and carbon dioxide [This study identified a number of aspects of athrooms , 8, 10.  dioxide . Bathroo dioxide .The tendency for resting to predominantly occur at low (< 0.75 m) to intermediate heights (0.75–1.5 m) was observed for both genera. At lower heights there is less air movement, and it is generally darker than the wall near the ceiling and often lit by light bulbs. Furthermore, ceiling fans are often installed, which may interfere with mosquito resting. Finally, at lower heights, there are often hanging objects such as clothes, towels and mosquito nets as well as furniture that create sheltered dark sites, offering an ideal hiding place to rest and digest . MosquitAe. aegypti were observed to increase in the living room in the afternoons, such rooms being the most juxtaposed to the outdoors. Such exiting behavior is thus likely important to take into account for similar studies.For both genera of mosquitoes, there was a tendency for numbers to decrease in the afternoon, suggesting that the mosquitoes were heading outdoors, potentially looking for oviposition sites. Oviposition has been found to peak in late afternoon to early evening . This miAe. aegypti in rural settings. The absence of eaves and low wind flow were associated with more Culex spp. in urban settings, although the strength of the associations observed was weak. In addition to such house characteristics, it was notable that infrequent use of fogging or larval control was associated with increased numbers of Culex spp. and Ae. aegypti. Whilst this would make sense, the subjective nature of memory on how frequently vector control was carried out may generate a bias and thus be treated with caution. Identifying household features that provide a conducive environment for mosquitoes can contribute to efforts for implementing push–pull strategies to make houses less attractive [Several of these explanations as to why the mosquitoes were thus distributed are supported by observed associations with the environmental features of the houses. Notably, hanging clothes at intermediate heights was associated with increased numbers of tractive . This cotractive . Whilst tractive , 28. SucComparing the performance of active aspiration versus passive sticky traps to measure mosquito abundance, we found that, broadly, both methods revealed the same resting behavior tendencies, suggesting that low-cost, low-manpower sticky traps offer a viable alternative to aspirators, even if their efficiency (mosquitoes per time) is lower.Ae. aegypti mosquitoes collected by aspiration in rural settings, indicating a protective effect by the presence of more geckos. On the other hand, Culex spp. were positively associated with geckos. This might suggest that the predation by geckos favored Culex spp. potentially through the intermediate predation of spiders, whereas Ae. aegypti was directly affected by gecko predation. However, these results only applied to the urban settings and require a more detailed study.Few have studied potential links between mosquito predators, food web interactions and mosquito vector control , 30. GecA limitation of our study was that we did not distinguish between mosquitoes resting on clothes compared to walls. This was due to methodological complications. However, it would be important for vector control strategies to know whether mosquitoes rest on clothes more than on walls. While it is possible to use IRS on walls, IRS cannot be done if mosquitoes rest on clothes. Furthermore, IRS in bathrooms is not likely to be effective, and other strategies should be employed in such situations. Another limitation of the study was the absence of meteorological data. However, as the villages were sampled sequentially and were taken into account in the analyses, the major findings of the work are unlikely to have been significantly altered by the inclusion of meteorological data. Furthermore, the results were so consistent across the sites whether using aspirators or sticky traps, that although the effect of changes in humidity, rainfall and temperature may have altered total numbers, the behavioral trends remain. Although data were analyzed separately by urban/rural setting, there might be heterogeneity within areas, especially urban areas, for example depending on rich and poor neighborhoods. We did not capture this potential urban heterogeneity. Another limitation is in the sampling strategy used for mechanical aspiration, which always started at the lower wall heights and then progressively moved upwards. Sampling might have been better designed to start at different heights in a randomly selected height protocol. However, that the mosquitoes were predominantly found resting at the lower heights would suggest that any disturbance of mosquitoes pushing them to higher heights did not happen. Finally, the gecko collections were not an initial aim of this study and would therefore benefit from a more targeted study informed by previous studies.Culex spp. are vectors of a number of arboviruses in the region, and thus our work provides an evidence base for understanding vector control for such vector-borne pathogens. Finally, this study highlights the importance of regular mosquito control, which can be targeted through education programs.Our work suggests that vector control using targeted IRS focusing on walls at heights lower than 1.5 m in bedrooms and bathrooms could be part of an integrated effective strategy for dengue vector control. Although we predominantly focused on dengue, Additional file 1: Table S1.Ae. aegypti mosquitoes collected by mechanical battery-driven aspirator differentiated by collection time, room and wall height above the floor in (A) rural areas and (B) urban areas in northeastern Thailand, 2019.Additional file 2: Table S2.Culex spp. mosquitoes collected by mechanical battery-driven aspirator differentiated by collection time, room and wall height above floor in (A) rural areas and (B) urban areas in northeastern Thailand, 2019.Additional file 3: Table S3.Ae. aegypti mosquitoes collected by sticky traps differentiated by collection room and wall height above floor in (A) rural areas and (B) urban areas in northeastern Thailand, 2019.Additional file 4: Table S4.Culex spp. mosquitoes collected by sticky traps differentiated by collection room and wall height above floor in (A) rural areas and (B) urban areas in northeastern Thailand, 2019.Additional file 5: Table S5. Household environmental and socioeconomic characteristics.Additional file 6: Table S6. Univariable P-values for association analyses of socioeconomic and environmental variables with Aedes aegypti abundance.Additional file 7: Table S7. Univariable P-values for association analyses of socioeconomic and environmental variables with Culex spp. abundance.Additional file 8: Dataset S1. Dataset on mosquito resting and gecko collections.Additional file 9: Dataset S2. Dataset on socioenvironmental characteristics.


Cytosolic DNA sensors (CDSs), expressed in various types of immune and tumor cells, recognize double-stranded DNA (dsDNA) in the cytoplasm. These molecules are activated after the dsDNA recognition and initiate a cascade of events that culminate in the activation of innate and acquired immunity. CDSs were previously believed to recognize pathogen-derived DNA only. However, they can also respond to cytosolic DNA derived from tumor cells. This suggests that CDSs can be used as small-molecule inhibitor targets in combination with existing immunotherapies to enhance anti-tumor immune responses. This review summarizes current research on the mechanisms underlying CDSs, absent in melanoma 2 (AIM2), cyclic GMP-AMP synthase (cGAS), and stimulator of interferon genes (STING)—downstream signaling effectors of cGAS—in the tumor microenvironment. Furthermore, this review discusses the prospects for future anti-tumor immunotherapy strategies based on these molecules.cGAS and AIM2 are CDSs that are activated in the presence of cytosolic dsDNA and are expressed in various cell types, including immune and tumor cells. The recognition of tumor-derived dsDNA by CDSs in the cytosol of tumor-infiltrating dendritic cells (TIDCs) activates the innate and acquired immunity, thereby enhancing anti-tumor immune responses. STING is the downstream signaling effector of cGAS that induces type I interferon (IFN) signaling. Owing to their ability to activate TIDCs, STING agonists have been intratumorally injected in several clinical trials to enhance the anti-tumor immune response elicited by immune checkpoint antibodies. However, they have shown minimal effect, suggesting the importance of optimizing the dose and route of administration for STING agonists and deciphering other immune pathways that contribute to anti-tumor immune responses. Recent studies have revealed that AIM2 activity induces pro-tumor growth through multiple parallel pathways, including inhibition of STING-type I IFN signaling. Thus, AIM2 could be a potential molecular target for cancer immunotherapies. This review summarizes the current research on the roles of cGAS, STING, and AIM2 in immune cells and tumor cells in the tumor microenvironment and discusses the future prospects of anti-tumor treatment approaches based on these molecules. Mammalian cells can sense pathogen invasion and induce innate immunity through nucleic acid recognition. When dsDNA enters the cytoplasm of dendritic cells (DCs) and macrophages after a bacterial or viral infection, CDSs are activated to induce an inflammatory response. These include cyclic GMP-AMP synthase (cGAS) and absent in melanoma 2 (AIM2).Vaccinia virus compared with 30% mortality in wild-type mice . cGAype mice . In addiype mice ,3,4.Listeria, Francisella, and Mycobacteria tuberculosis. The AIM2-deficient mice are more susceptible to these intracellular bacteria .,23.22,23Most studies focus on immune cells within tumors. Therefore, data on the function of STING in tumor cells are limited. Studies using mouse and human colon cancer samples, as well as lung cancer model mice and the TCGA lung cancer dataset, found that tumor cells suppress the high-level expression of cGAS and STING through epigenetic regulation or the production of DNase ,25. ConsChromosomal instability (CIN) is a phenomenon that has been observed in some cancer types, where some or all chromosomes increase or decrease in number. Binding between cGAS and dsDNA is more likely to occur in “CIN-high” cancers than in “CIN-low” cancers. Although STING is activated, the downstream type I IFN signaling pathway is not, resulting in discrepancies, such as a high potential for cancer cell proliferation and metastasis . In CIN-This finding suggests that the activation of the type I IFN signaling pathway could be suppressed despite the activation of tumor cell STING signals. Therefore, the treatment should cause the release of cGAMP and CDNs from tumor cells into the tumor’s microenvironment to activate STING in the cDC1 TIDCs that have infiltrated the cancerous growth, thereby enhancing the anti-tumor immune response.The first STING agonist evaluated for efficacy against cancer was DMXAA, which exhibited antitumor activity in preclinical models. However, phase III trials that compared DMXAA in combination with carboplatin and paclitaxel chemotherapies (CP therapy) to only CP therapy for previously untreated non-small cell lung cancer failed to demonstrate an improved response rate, progression-free survival (PFS), or OS . Later sBased on previous results of DMXAA, Aduro Biotech has developed MIW815 (ADU-S100), a CDN that agonizes cGAMP, thereby activating human STING isoforms and acting as a human STING agonist. CDNs are susceptible to enzymatic degradation by serum phosphatases and hydrolases , leading+CD8− dendritic cells but not on cDC1s in tumor-draining lymph nodes. In contrast, oral MSA-2 significantly decreased the tumor volume of melanoma, colon, and lung cancer mouse models when combined with anti-PD-1 therapy. These results suggest that the molecular properties of STING agonists and administration modes should be optimized to achieve optimal treatment outcomes.To overcome these challenges, a non-CDN small molecule STING agonist, GSK3745417 (diABZI), that is systemically administered, has been developed . As it sNew types of packaged STING-activating CDNs with improved cytosolic delivery and stability are also candidates for future STING agonists . Since l+ T-cells. In addition, loss of AIM2-dependent IL-1β and IL-18 processing further enhances the treatment response by limiting the recruitment of T regulatory cells (Tregs). Hence, AIM2-deficient DC vaccination not only enhances immune responses to the tumor by activating STING but also suppresses IL-1β and IL-18 production, resulting in synergistic therapeutic responses . Tumor cells that are bonded to antibodies (molecularly targeted drugs), such as the anti-human epidermal growth factor receptor 2 (anti-HER2) and anti-CD20 antibodies, are taken up into TAMs as a result of antibody-dependent cellular phagocytosis (ADCP). After ADCP, TAMs inhibit NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and T-cell-mediated cytotoxicity in a breast cancer and lymphoma mouse model. Moreover, AIM2 is recruited to the phagosomes through Fcγ receptor signaling and activated by sensing the phagocytosed tumor DNAs through the disrupted phagosome membrane. This subsequently activates IL-1β signaling, which induces the expression of the immune checkpoint molecules PD-L1 and indoleamine 2,3-dioxygenase (IDO) on the TAM cell surface. These results revealed the role of ADCP of TAMs in cancer immunosuppression and suggested that simultaneous therapeutic antibodies and AIM2 inhibition of TAMs provide synergistic effects in cancer treatment .c-myc and, conversely, suppresses the expression of FOXP3. Therefore, when AIM2 is removed from the Tregs at sites of chronic inflammation, the AKT–mTOR pathway is activated, c-myc expression is increased, and FOXP3 expression is decreased. This reduces the number of bona fide Tregs. Moreover, multiple models of chronic inflammation have shown that increasing the amount of IFN-γ produced by Tregs and decreasing the immunosuppressive action exacerbates inflammation. These findings suggest that, in addition to its suppression in TIDCs, the suppression of AIM2 in TAMs and Tregs also increases antitumor immune response [Although AIM2 has long been investigated for its function in bone marrow cells, such as DCs and macrophages, it is more strongly expressed in Tregs. Therefore, there has been a new focus on its function in this class of T-cells. In experimental autoimmune encephalomyelitis and chronic inflammation in enteritis, the expression of AIM2 by Tregs maintains the expression of the transcription factor forkhead box P3 (FOXP3). In other words, AIM2 maintains Treg homeostasis in cases of inflammation. As AIM2 binds with the receptor for activated C kinase 1 (RACK1) and protein phosphatase 2A (PP2A), it suppresses activation of the protein kinase B–mammalian target of the rapamycin (AKT–mTOR) pathway. This signaling pathway promotes the expression of the cancer gene response .AIM2 is hardly expressed in melanomas and was initially discovered as a tumor suppressor gene . Most meThe functions of AIM2 have been investigated in many cancer types, including melanoma. For instance, AIM2 has long been known as a marker for poor prognosis in colorectal cancer. This CDS suppresses the phosphoinositide 3-kinase (PI3K)–AKT signaling pathway in colorectal cancer cells. In patients with colorectal cancer with low AIM2 expression, the PI3K–AKT signaling pathway is upregulated, which increases the proliferative and survival abilities of the cancer cells, making them highly malignant. In addition, research using a colon cancer mouse model revealed that low AIM2 expression induced an imbalance of the intestinal flora (dysbiosis), thereby providing a suitable environment for the proliferation of colorectal cancer cells . SimilarSTING agonists that activate cDC1 TIDCs have been investigated as adjuvants for increasing the antitumor immune response elicited by anti-PD-1 immunotherapy. However, the results of combination therapy with anti-PD-1 antibodies + intratumoral STING agonist administration have not been promising. Therefore, STING agonists that can be administered systemically and stabilize the close confirmation of STING to decrease the toxicity may be an effective approach for targeting the STING-type I IFN signaling pathway. Combination therapy consisting of the anti-PD-1 Ab+ systemic STING agonist should be investigated in future clinical trials since it has the potential to transform the therapeutic landscape once optimized.+ T-cells. As there is no drug that selectively inhibits AIM2, it would be impossible for a treatment that targets this molecule to be put to clinical use in the near future. In addition to being used as a STING agonist, anti-AIM2 therapy would activate TIDCs and act as an IL-1β and IL-18 inhibitor, thus acting on more pathways than the STING agonist does. Therefore, as a candidate for molecularly targeted drug therapy, AIM2 could be more effective at reinforcing antitumor immunotherapies. Researchers have recently reported the engineering of a nanomolecular STING agonist vaccine that has been modified to be taken up by cDC1 [+ cells could be a novel cancer immunotherapy.In addition, there remains a need to analyze other immune pathways that contribute to the immune infiltration into tumors and thus help to determine their “hot” or “cold” states. Researchers have begun testing antibodies against TIM-3, which is an immune checkpoint receptor that suppresses the efficacy of STING agonists and regulates CD8 by cDC1 . An AIM2